Abstract
Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ROL) can be oxidized to all-trans-retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-γ (RARγ) knockout ESC lines generated by CRISPR/Cas9 and homologous recombination, we demonstrate that EtOH signals via RARγ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 (Hoxa1) and cytochrome P450 family 26 subfamily A member 1 (Cyp26a1) transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of Hoxa1 and Cyp26a1 requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene Rdh10 and a functional RARE in the ROL transporter stimulated by retinoic acid 6 (Stra6) gene are required for EtOH induction of Hoxa1 and Cyp26a1 We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RARγ-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation.
Highlights
Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood
chromatin immunoprecipitation (ChIP) assays, and retinoic acid receptor-␥ (RAR␥) knockout embryonic stem cell (ESC) lines generated by CRISPR/ Cas9 and homologous recombination, we demonstrate that EtOH signals via RAR␥ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers
We demonstrated a reduction in pluripotency in ESCs upon EtOH treatment for 96 h, as EtOH decreased staining intensity compared with 0.1% DMSOtreated cells by 7.6% (p ϭ 0.020) (Fig. S1A)
Summary
Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ ROL) can be oxidized to all-trans-retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH’s effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-␥ (RAR␥) knockout ESC lines generated by CRISPR/ Cas and homologous recombination, we demonstrate that EtOH signals via RAR␥ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Embryos of EtOH-treated mice show higher RA levels in specific sites [15], prompting us to explore the mechanisms underlying the effects of EtOH on RAR signaling in embryonic stem cells (ESCs) in greater depth
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